The present invention relates to a regulatory region that controls expression of a gene. In particular, it relates to the nucleotide sequence of a 5xe2x80x2 regulatory region and fragments thereof, which promote transcription of the myostatin gene, to be referred to herein as the myostatin gene promoter. In particular, it relates to the 5xe2x80x2 regulatory region and fragments thereof, which promote transcription of the murine myostatin gene. The invention also relates to methods of using this region to regulate expression of a heterologous gene in cells or animals, to engineer host cells, to screen for compounds that activate or inhibit its transcription and expression, as well as methods of inhibiting its expression in cells for the promotion of muscle growth in livestock, poultry, fish and companion animals, as well as for the treatment of muscle wasting and neuromuscular diseases.
Growth and differentiation factor-8 (GDF-8 or myostatin), which will be referred to herein as myostatin, is a member of the transforming growth factor-xcex2 (TGF-xcex2) superfamily of secreted growth and differentiation factors (McPherron, et al. Nature 387:83 (1997)). The members of the TGF-xcex2 family are synthesized as large precursor proteins that are proteolytically cleaved at a cluster of basic residues approximately 110-140 amino acids from the C-terminus. The C-terminal regions, which are active as disulfide-linked dimers, have some level of amino acid sequence and structural similarity. The TGF-xcex2 family is characterized by a consensus amino acid sequence and conserved cysteine residues involved in intrachain disulfide bonds that result in a cystine knot protein fold. Members of the family include Mullerian inhibitory substance (MIS) (Behringer, et al., 1990, Nature, 345:167), bone morphogenic proteins (BMPs) (Sampath, et al., 1990, J.Biol.Chem. 265:13198), growth and differentiation factors (GDFs), inhibins, and Drosophila decapentaplegic protein (DPP-C) (Padgett, et. al., 1987, Nature 325:81).
Myostatin has been identified in all vertebrates examined, including mouse, rat, human, baboon, cattle, pig, sheep, chicken, turkey, cats, dogs and fish. A disruption of the myostatin gene in mice leads to large increases in skeletal muscle mass resulting from muscle hyperplasia and hypertrophy and is at least partially responsible for the hypermuscled phenotype of mice homozygous for the Cmpt (compact) mutation (McPherron et al. (1997); Szabo, G. et al. Mammalian Genome 9:671 (1998)). In double-muscled breeds of cattle such as the Belgian Blue or Piedmontese, the bovine myostatin gene has been shown to be partially deleted or contains a missense mutation. (Grobet, L. et al. Nat. Genet. 17:71 (1997); Kambadur, R. et al, Genome Res. 7:910 (1997); McPherron and Lee, Proc. Natl. Acad. Sci USA 94:12457 (1997)). Thus, myostatin seems to function as a negative regulator of skeletal muscle growth, however the mechanisms that regulate its expression have not been described (McPherron et al. (1997)).
The sequence of the cDNA for myostatin has been determined for each of these species and the deduced amino acid sequence is extraordinarily conserved, particularly the amino acid sequence of the C-terminal 109 amino acids. The nucleotide sequences of the full-length amino acid coding regions of the human and murine myostatin genes were disclosed in WO94/21681 to Lee and McPherron (1994). The nucleotide sequences of the full-length amino acid coding regions of the rat and chicken myostatin genes were disclosed in WO 98/33887 to Lee and McPherron (1998). While the coding sequences of the human and murine myostatin genes have been determined, those regulatory sequences which control expression of these genes remain uncharacterized.
The present invention relates to a transcription regulatory region of a myostatin gene. In particular, the invention relates to a 2.5 kb polynucleotide sequence immediately 5xe2x80x2 to the murine myostatin coding sequence, its nucleotide sequence and methods of using this regulatory region, and fragments thereof. The present invention relates to the use of the myostatin promoter of the present invention to direct expression of a target gene in a cell or tissue specific manner, e.g., muscle cells or tissue. The present invention relates to the use of the myostatin promoter of the present invention in high throughput screens to identify test compounds which inhibit myostatin promoter activity or myostatin expression. The present invention relates to methods for increasing muscle mass and feed efficiency of livestock, poultry or fish, in particular to engineer animals with increased lean meat in order to decrease the time required to bring the animals to slaughter. In accordance with the present invention, inhibitors of the myostatin promoter may be used to inhibit myostatin expression as a method of treating disorders related to expression of the myostatin gene, such as muscle wasting associated with aging or disease in humans and companion animals.
The invention is based on the determination of the complete nucleotide sequence of the 2.5 kb murine myostatin gene promoter. Although the nucleotide sequence of the myostatin gene was previously reported, the myostatin promoter region has not yet been reported. Using a combination of manipulations, the promoter region of the murine myostatin gene was identified, sequenced and reported herein. When the 2.5 kb region and certain fragments thereof were placed upstream of a luciferase reporter gene in an expression vector, and introduced into muscle cell lines, these sequences induced the expression of the reporter gene. In addition, the identified promoter sequence of the murine myostatin gene and the identified promoter sequence of the porcine myostatin gene show a high degree of homology.
It is an object of the invention that the 2.5 kb regulatory region, or transcriptionally active fragments thereof be inserted in an expression vector to regulate the expression of a downstream coding sequence in a cell in vitro and in vivo.
In another embodiment of the invention, the aforementioned vector is stably integrated into the genome of a host cell. The cell is treated with a test compound in a screening assay for determining the ability of the compound to activate or inhibit the transcriptional activities of the regulatory region. The selected compounds may be formulated as pharmaceutical compositions for the promotion of muscle growth or for treatment of disorders related to aberrant expression of myostatin.
In yet another embodiment of the invention, the aforementioned vector is introduced into an embryonic cell or other type of cell, for the construction of a transgenic animal to regulate the expression of a transgene in a tissue specific manner.
It is also an object of the invention that polynucleotides complementary to the 2.5 Kb regulatory region or portions thereof be delivered to cells to inhibit the transcription activities of the endogenous 2.5 Kb region, thereby downregulating the expression of myostatin. Such polynucleotides are useful for the promotion of growth, or in the treatment of diseases associated with myostatin expression, such as muscle wasting and neuromuscular disease.
The present invention also relates to methods of treating livestock, poultry or fish to promote muscle growth. In particular, the invention relates to methods for increasing muscle mass and feed efficiency in order to increase growth so that animals can be brought to slaughter sooner. The methods of treatment of the present invention are applicable to humans and non-humans.
The invention further relates to methods for the treatment of disorders, such as muscle wasting, neuromuscular disease, cancer and aging, wherein such methods comprise administering compounds which modulate the expression of a myostatin gene so symptoms of the disorder are ameliorated.
In addition, the present invention is directed to methods that utilize the myostatin promoter sequences for the diagnostic evaluation, genetic testing and prognosis of a disease or disorder associated with myostatin expression.
The invention still further relates to methods of identifying compounds capable of modulating the activity of myostatin promoters and the expression of myostatin genes, wherein such methods comprise administering a compound to a cell that expresses a gene under the control of a myostatin promoter or a transcriptionally active fragment thereof, measuring the level of gene expression or gene product activity and comparing this level to the level of gene expression or gene product activity produced by the cell in the absence of the compound, such that if the level obtained in the presence of the compound differs from that obtained in its absence, a compound capable of modulating the expression of the myostatin gene or promoter activity has been identified.